Historically, chemical analysis of charged analytes has been carried out using slow, expensive, low resolution, and solvent, gas, and instrumentation intensive techniques such as ion-exchange chromatography, atomic absorption and inductively coupled plasma emission spectroscopy.
More recently, capillary electrophoresis has become an important tool for chemical analysis of charged analytes as well as a wide variety of other separation problems. The introduction and development of capillaries, small-diameter chromatographic columns containing an internal stationary phase, have played an important part in the advancement of many areas of science and industry.
In capillary zone electrophoresis (CZE) analyte components move from an injector end of a capillary to a detector end of a capillary under the influence a voltage difference applied across the length of the capillary. Because of the different mass-to-charge ratios (m/z) of analyte components, the individual components of the analyte move through the column with different velocities. This difference in movement through the capillary leads to the physical separation of the components of the analyte into individual electrophoretic zones. The separated components of the analyte are then detected instrumentally as they are eluted from the capillary.
Advantages of CZE include a high theoretical plate number, about 3-10 times higher than other analytical techniques such as high performance liquid chromatography (HPLC) owing to the band moving in a flat rather than a parabolic profile, no mechanical pump, rapid analysis time, low cost per test, low solvent requirements, full automation, and ability to modify the selectivity of the separation simply by adding additives to the buffer to alter the velocity of some analytes.
However, disadvantages of CZE include poor sensitivity of absorbency detection and problems with sample matrix and sample adsorption to the capillary. Many of these issues are the direct result of the current method of manufacturing the capillary columns. Currently polymerization of the internal stationary phase is initiated external from the capillary resulting in an inconsistent stationary phase profile, limiting capillary length, sensitivity, and resolution. Enhancing these characteristics of the capillary would be a great advance in chromatography.
Capillary zone electrophoresis is an attractive alternative to liquid chromatography, particularly in proteomics research. CZE provides rapid and efficient separation of biological molecules. Uncoated capillaries generate high electroosmotic flow that leads to rapid separations, but with a short separation window that limits the peptide and protein identification in analysis of complex proteomes. Instead, proteomic analysis by CZE requires the use of capillaries that have had their interior uniformly coated with a polymer, which both suppresses electroosmotic flow and minimizes peptide interaction with the wall.
A capillary coating of linear polyacrylamide (LPA) is commonly used in CZE separations. Typically, the LPA coating is produced by covalently bonding acrylamide monomer to the inner wall of the capillary. The capillary is first treated with a vinyl-silane to covalently graft a double bond to the silica capillary wall. Next, a polymerization mixture is prepared by mixing a monomer (e.g., acrylamide), polymerization initiator (e.g., ammonium persulfate (APS)), and water in a tube, external to the capillary. The solution is then degassed by N2(g) to remove oxygen. Tetramethylethylenediamine (TEMED) is then added as a catalyst to the mixture and the polymerization reaction commences. The polymerizing mixture must then rapidly be introduced into the pretreated capillary. The time between the addition of TEMED and introduction of the mixture to the capillary critically affects the performance and reproducibility of the capillary. In addition, a special device is typically required to perform the step of introducing the polymerizing mixture to the capillary. This method results in a capillary coating that is not uniform throughout the capillary and exposed column surface, which lacks uniformity from batch to batch, has limited capillary length, and poor capillary resolution and reproducibility.
Accordingly, there is a need for a capillary with improved coating uniformity, reproducibility, resolution, and higher separation efficiency. Additionally, there is a need for such a capillary capable of being fabricated in long columns/with increased length compared to currently available capillaries. The present invention describes a novel method of producing a capillary that provides all of the above-mentioned desirable characteristics through a simple procedure carried out under mild thermal conditions.